The present invention relates to the separation of biological compounds or biomolecules utilizing reverse-phase chromatographic media.
Biological compounds, such as proteins, have become important in a variety of research or analytical applications, as well as commercial applications, such as drug candidates for various therapeutic uses. One of the greatest challenges lies in the development of cost effective and efficient processes for separation of such biological materials. While many methods are now available for separation of such materials, crude products contain not only the desired product but also closely related impurities that are difficult to separate from the desired product. Moreover, biological sources of these products usually include complex mixtures of materials that include various target compounds that are present in small amounts, and may also vary in size and shape.
Generally, biological materials are produced by cell culture, using either mammalian or bacterial strains engineered to produce the materials of the interest by insertion of a recombinant plasmid containing the gene for that protein. Since the strains used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids and growth factors that are usually supplied from preparations of animal serum. Separation of the desired biomolecule from the mixture of compounds fed to the cells and from the byproducts from the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge. Usually, the separation procedure is multi-step requiring expensive apparatus and chromatographic media.
Procedures for purification of biomolecules from cell debris initially depend on the sight of expression of the biomolecule. Some biomolecules may be secreted directly from the cell into the surrounding growth media, while others are made intra-cellularly. For the latter biomolecules, the first step of a purification process involves lysing or destruction of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such destruction releases the entire contents of the cell into the homogenate, and in addition produces sub-cellular fragments that are difficult to remove due to their small size. Usually, these are removed by differential centrifugation or by filtration.
After a clarified solution containing the biomolecule of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of different chromatography techniques. These techniques separate mixtures of biomolecules on the basis of their charge, degree of hydrophobicity, or size. Several different chromatography media are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular biomolecule involved. Affinity chromatography, which exploits a specific interaction between the biomolecules to be purified in a second biomolecule, such as a specific antibody, may also be a separation technique for some biomolecules.
The essence of each of the separation methods is that biomolecules can be caused either to move at different rates through chromatographic media, achieving a physical separation that increases as they pass further through the media, or to adhere selectively to the separation media, being then differentially eluted by different solvents. In some cases, the desired biomolecule is separated from impurities when the impurities specifically adhere to the media, and the biomolecule of interest does not.
The major performance measures of chromatography techniques are productivity and peak resolution. Productivity refers to specific throughput. It is a measure of the mass of solute that can be processed per unit volume of chromatography matrix. Generally, productivity improves with increases in the surface area per unit volume of the matrix, the rate of solute mass transfer to the sorbent surface, the rate of adsorption and desorption, and the fluid flow velocity through the matrix. Resolution is a measure of the degree of purification that a system can achieve. It is specified by the difference in affinity among solutes in the mixture to be separated and by the systems inherent tendency towards dispersion or band spreading. Affinity of the solutes is controlled by the nature in the process liquid and the chemical properties of the chromatography media. Band spreading is controlled primarily by the geometry of the chromatographic media (e.g. the surface area, particle size, etc.) and the mass transfer rates from the solute to the media surface during the chromatographic procedure.
The application of modern liquid chromatographic techniques by high performance liquid chromatographic (HPLC) has led to an improvement of separation, characterization, and purification of biomolecules. Liquid chromatographic using a reverse phase packing has been found to be an effective tool in both qualitative and quantitative analysis for biological substances in blood, serum, or plasma. Typically, the reverse phase packing material is made up of bonding alkyl groups to porous inorganic oxides, e.g., silica, and most typically the packing is a pore silica having octadecylsilane bonded to it. The porous silica particles are available in a variety of forms, with different sizes of particle and pore size within the particle. The size of particle chiefly determines the packing properties of the material, which determine the rate of flow and the backpressure when the material is used as a column. The pore size, however, determines the size of biological material that has access to the interior of the pore. Typically, pore sizes vary from 100 to 500 Angstroms in size. The size of the particle may depend upon the nature of the separation process. A mixture of water and organic solvent are used to elute molecules of interest. Biomolecules are often loaded under highly aqueous conditions to maximize their binding to the reversed-phase material. These molecules will elute at the threshold organic solvent concentration. One means for their elution involves a gradient separation whereby the organic solvent content is increased per unit time.
In analytical chromatography, peak resolution is of paramount importance and efforts have been made to minimize band spreading and maximize peak resolution. Efforts have also been made to increase the speed of separation of biomolecules in order to allow for expedited analysis that is needed to conduct competitive research in proteomics. Higher flow rates have been utilized to achieve higher speed of biomolecule separation, but such processes run at much higher pressures (e.g., above 5,000 psi) that cannot be implemented in standard HPLC equipment.
There have been numerous efforts of utilizing inorganic porous media to separate biological materials using high performance liquid chromatography (HPLC). See for example U.S. Pat. Nos. 4,289,690 and 4,959,340. Additionally, U.S. Pat. No. 5,451,660 describes the use of reverse phase high-pressure liquid chromatography for the purification of polypeptides. In such a process, the different components of the mixture introduced into the column possess different respective degrees of solubility in the stationary phase (chromatographic media) and in the mobile phase (the mixture passing through the column). As the mobile phase flows over the stationary phase, there is an equilibrium in which the sample components are partitioned between the stationary phase and the mobile phase. As the mobile phase passes through the column, the equilibrium is constantly shifted in favor of the mobile phase. This occurs because the equilibrium mixture, at any time, is exposed to fresh mobile phase and partitions into the fresh mobile phase. As the mobile phase is carried down the column, the mobile phase is exposed to fresh stationary phase and partitions into the stationary phase. A separation of a mixture of components occurs because the mixture of components has slightly differently affinities for the stationary phase and/or solubilities in the mobile phase, and therefore, has different partition equilibrium values. Thus, the mixture of components passed down the column at different rates.
In U.S. Pat. No. 5,585,236, ion pair reverse-phase high-pressure liquid chromatography is described as a process for separation DNA using a non-poly separation media, wherein the process utilizes a counter-ion agent and an organic solvent to release the DNA from the separation media. More recently, analysis and separation of RNA molecules has been performed using matched ion polynucleotide chromatography. See U.S. Pat. No. 6,475,388 B1.
Profusive chromatography has been utilized to increase the efficiency in chromatographic separations by increasing the surface area of the chromatographic media and the fluid flow velocity through the media in order to increase the liquid through the column. See U.S. Pat. No. 5,833,861. This is accomplished using chromatographic media possessing a first pore set having a great mean diameter than the members of a second pore set.
Heretofore, there has been no HPLC process that provides for efficient separation of biological substances combined with the advantages of high speed of biomolecule separation. Therefore, there is a need for a separation process for such biological materials that allows for high separation speed as well as increased recovery of the target biological material that may be utilized in analytical, preparative and process chromatographic applications.